Design, synthesis, and binding affinities of potential positron emission tomography (PET) ligands with optimal lipophilicity for brain imaging of the dopamine D3 receptor. Part II

Article abstract of DOI:10.1016/j.bmc.2008.11.044

In the search for compounds with potential for development as positron emission tomography radioligands for brain D₃ receptor imaging, a series of N-[4-(4-arylpiperazin-1-yl)butyl]arylcarboxamides with appropriate lipophilicity (2 < log P < 3.5) were synthesized and tested in vitro. Some of the final compounds showed moderate-to-high dopamine D₃ receptor affinities but lacked selectivity over D₂ receptors.

Full text of DOI:10.1016/j.bmc.2008.11.044

Bioorganic & Medicinal Chemistry 17 (2009) 758–766
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and binding affinities of potential positron emission
tomography (PET) ligands with optimal lipophilicity for brain imaging
of the dopamine D3 receptor. Part II
*
Marcello Leopoldo , Enza Lacivita, Paola De Giorgio, Marialessandra Contino,
Francesco Berardi, Roberto Perrone
Università degli Studi di Bari, Dipartimento Farmaco-Chimico, via Orabona, 4, 70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search for compounds with potential for development as positron emission tomography radioli-
gands for brain D3 receptor imaging, a series of N-[4-(4-arylpiperazin-1-yl)butyl]arylcarboxamides with
appropriate lipophilicity (2 < logP < 3.5) were synthesized and tested in vitro. Some of the final com-
pounds showed moderate-to-high dopamine D3 receptor affinities but lacked selectivity over D2
receptors.
Received 9 September 2008
Revised 13 November 2008
Accepted 15 November 2008
Available online 24 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
D3 receptors
Positron emission tomography
Structure–affinity relationships
Arylpiperazine
Lipophilicity
1. Introduction
tor antagonists are efficacious in animal models of cocaine-,
nicotine-, alcohol-, and heroin-seeking behaviors.14 The D2 and
Positron emission tomography (PET) is a powerful in vivo imag-
ing technique that is showing its potential in various areas such as
diagnosis, drug discovery, and target validation. This functional,
nuclear imaging technique can trace the fate of radiolabeled mole-
cules directly, but non-invasively, and allow precise pharmacoki-
netic and pharmacodynamic measurements. Molecular imaging
provides unique data that can aid in selecting the best drug candi-
dates, determining optimal dosing regimens, clearing regulatory
hurdles and lowering risks of failure.1 Development of suitable
radioligands has allowed the visualization of several molecular tar-
D3 dopamine receptors have approximately 46% amino acid
homology. However, the transmembrane spanning regions of the
D2 and D3 receptors, which are thought to construct the ligand
binding site, share 78% homology.15 Because of the high degree
of homology, it has been difficult to obtain compounds that can
bind selectively to either the D2 or the D3 dopamine receptors.16,17
Various dopamine D3 receptor ligands have been labeled with a
positron emitter (compounds 1–8, Table 1), but none of them
was suitable for in vivo imaging of D3 receptors.18–24 An adequate
PET radioligand should fulfill a number of requirements.25 The can-
didate radioligand should be suitable for high specific activity
labeling with carbon-11 (half-life = 20.38 min) or fluorine-18
(half-life = 109.8 min). The tracer needs to have high affinity for
the target receptor. In particular, it is preferable that the Bmax
clearly exceeds the Kd of the ligands (ideally Bmax/Kd > 10). Further-
more, the selectivity over other receptors needs to be good (about
100-fold). It should not be toxic. It should cross the blood–brain
barrier. This implies an appropriate lipophilicity (logP = 1.5–4),
low molecular weight (<450 Da), and absence of P-glycoprotein-
mediated-efflux of the tracer. On the other hand, the lipophilicity
should not be too high in order to avoid non-specific binding. This
is an essentially non-saturable component of the total tissue up-
take of a radioligand, usually attributed to adhesion to protein
and lipids. Therefore, it appears that there is an optimal range of
lipophilicity for brain radioligands, wherein brain uptake is high
gets into the central nervous system (CNS), including monoamine
4
transporters (NET, SERT, DAT),2 dopamine D2,3 serotonin 5-HT1A
,
mGLU5,5 opioid,6 and cannabinoid CB1 receptors.7 Also the dopa-
mine D3 receptor subtype has been the target for PET tracer devel-
opment. Molecular genetic studies of G-protein coupled receptors
have defined two families of dopamine receptors, the D1-like (D1
and D5 receptor subtypes) and D2-like (D2, D3, and D4 receptor sub-
types) receptors based upon structural and pharmacological simi-
larities.8,9 Various pharmacological studies have investigated the
D3 receptor as an interesting therapeutic target for the treatment
of schizophrenia,10,11 Parkinson’s disease,12 drug-induced dyskine-
sia.13 Recent studies have shown that selective dopamine D3 recep-
* Corresponding author. Tel.: +39 080 5442798; fax: +39 080 5442231.
E-mail address: leopoldo@farmchim.uniba.it (M. Leopoldo).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.044

M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
759
Table 1
Structures and properties of reported radiolabeled dopamine D3 ligands
Compound
Structure
D3 affinity (Ki, nM)
Calculated logP valuesa
Ref.
18
O
O
N
N
1 (BP-897)
1.4
4.52
N
N
O
N
H
O
Cl
2
0.6
6.10
5.72
5.37
3.88
4.98
19
19
20
21
22
Cl
N
H
O
N
Cl
3
0.13
0.23
0.56
1.1
N
Cl
O
O
N
H
O
N
4 (FAUC346)
5 (2-MMC)
6
N
O
N
H
S
O
O
N
N
O
N
H
O
N
Cl
N
Cl
N
H
F
S
N
N
7 (RGH-1756)
0.12
1.3
3.71
5.40
23
24
N
N
N
O
O
O
S
8 (GR218231)
O
O
N
a
Calculated with ACD/Labs 7.0 (Advanced Chemistry Development, Inc., Toronto, ON, Canada).
and non-specific binding comparatively weak. From literature data
a value of logP = 3.5 appears to be the acceptable upper limit of
lipophilicity for a PET radioligand.26 Finally, the radioligand metab-
olism should not produce radiolabeled metabolites that enter the
brain. Clearly, these requirements might be conflicting, inasmuch
as many chemical and pharmacologic parameters are associated
with in vivo behaviors that might affect the final image qualities
in opposite directions. Dopamine D3 receptor ligands 1–8 pos-
sessed several of the above mentioned features. In fact, they dis-
played nanomolar to subnanomolar affinity for D3 receptors
ranging from 0.12 to 86 nM and D2/D3 Ki ratios ranging from 4.9
to >5000. However, they failed to visualize dopamine D3 receptor
in vivo due to a low signal for specific binding to the D3 receptor
(compounds 1–5), disappointing binding characteristics in preli-
minary autoradiography experiments (compound 6), D3 affinity
at in vivo conditions not sufficient to visualize the D3 receptors
in the brain (compound 7), or interaction with another molecular
target (compound 8). Thus, the discovery of highly selective radio-
ligands as PET tracers is still an important task to highlight the
pathophysiological role of the D3 receptor. In a previous paper
we have described a series of potential PET radioligand for the
visualization of brain dopamine D3 receptors.27 That series origi-
nated from the high affinity D3 receptor ligand 3 through structural
modifications targeted to lower lipophilicity and to leave un-
changed the high affinity for D3 receptor. A significant reduction
in lipophilicity was achieved essentially by substituting the 2,3-
dichlorophenyl group. However, this moiety revealed to be essen-
tial for high affinity and selectivity for D3 receptor. Nonetheless, we

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M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
identified derivatives 9a,b (Table 2) which displayed good D3
receptor affinities (Ki values 5.4 and 3.8 nM, respectively) and lipo-
philicity values within the optimal range (clogP = 3.05 and 2.76,
respectively). However, due to their modest selectivity over D2
receptors (10-fold), we were forced to undertake structural modi-
fications of 9a,b with the aim to increase the specificity for D3
receptor.
partitioning of a compound between octanol and aqueous solution
(logP). Terms commonly associated with the various methods of
lipophilicity assessment include logP, logP(oct), logP(app), logD,
clogP, DlogP, logKw, PC, logKw, logP (hex), logP 7.4, logP 7.2,
log Oct/water, as well as many others. There appears to be a lack
of standardization regarding the methods and terms used, perhaps
explaining why different optimal ranges of drug lipophilicity can
be found throughout the literature. When lipophilicity is expressed
as logP (partitioning of the neutral molecule species) or logD7.4
(partitioning of all species present in solution at a given pH and
therefore accounting for solubility effects associated with ioniza-
tion), compounds that seem most effective for imaging have logP
or logD7.4 < 3.5. On the basis of such considerations, we modified
our references 9a,b by designing compounds 10a,b–15a,b which
showed computer estimated values of lipophilicity below the
guideline value (3.5). The logP, logD7.4 and pKa values of the final
compounds were experimentally determined by potentiometric
titration (Table 2), because calculated values are normally referred
to the neutral (and more lipophilic) species only, and therefore
might differ from those obtained experimentally.
Experimental logP values of target compounds did not differ
greatly from clogP values. The most notable differences were ob-
served for compounds 12b and 13a (0.43 and 0.38 logarithmic unit
differences, respectively). Therefore, compounds 9a,b–15a,b pre-
sented lipophilicity within the optimal range. Considering the
pKa values of target compounds, it can be deduced that the per-
centage of protonated species at physiological pH is not very high,
and this accounts for the slight differences between logP and
logD7.4 values. Moreover, the basicity of the nitrogen of the piper-
azine ring depended upon the structural feature of the compounds.
In particular, the 2-substituted derivatives 9a,b and 13a,b showed
the highest pKa values that reflected in a more marked lowering of
logD7.4 values. On the other hand, the less pronounced difference
between logP and logD7.4 value was observed for compound 15b
2. Chemistry
The synthesis of the target compounds (Scheme 1) started from
the 1-arylpiperazines 17–22. Among them, only 20 was not com-
mercially available. Therefore the following synthetic pathway
was devised: 1-bromo-3,5-difluorobenzene reacted with sodium
methoxide to give the methoxy derivative 16 which underwent
Buchwald–Hartwig cross-coupling reaction to give the piperazine
20. Next, 1-arylpiperazines 17–22 were alkylated with the appro-
priate
x-chloroalkylnitrile, affording nitriles 23–28. Reduction of
the cyano group of 23–28 by borane methyl sulfide complex
yielded the key amines 29–32 and hydroxyphenyl derivatives 33
and 34. These latter were first protected with BOC then were re-
acted with 2-fluoroethylmesylate28 (compounds 35 and 36) and
subsequently deprotected to give the key amines 37 and 38. The fi-
nal compounds 9a,b–15a,b were prepared by condensing amines
29–32 and 37–38 with the appropriate carboxylic acid.
3. Results and discussion
3.1. Lipophilicity evaluation
The pivotal role of PET tracer lipophilicity is well recognized and
it has been reviewed in depth by Waterhouse.26 Lipophilicity can
be measured in various theoretical and experimental ways. The
most common experimental lipophilicity measurement involves
Table 2
Physicochemical properties and binding affinities of target compounds 9a,b–15a,b
R
N
N
H
N
O
a: Ar =
b: Ar =
N
Ar
N
N
( )n
N
O
c
Compound
n
R
clogPb
logP
logD7.4
pKa
Ki, nM SEMa
D2
51.7 3.7
D3
4.23 1.20
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
Haloperidol
Quinpirole
4
4
3
3
4
4
4
4
4
4
4
4
4
4
2-OCH3
2-OCH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
2-OCH2CH2F
2-OCH2CH2F
3-OCH2CH2F
3-OCH2CH2F
3-F,5-OCH3
3-F,5-OCH3
3.05
2.76
2.75
2.46
2.93
2.64
2.84
2.55
3.28
2.99
3.16
2.87
3.33
3.04
3.10 0.035
2.79 0.021
2.59 0.026
2.66 0.020
2.93 0.016
2.80 0.038
2.80 0.037
2.77 0.036
2.90 0.030
2.79 0.012
2.90 0.011
2.87 0.011
3.16 0.016
3.34 0.013
2.39
2.26
2.22
2.34
2.51
2.48
2.37
2.41
2.40
2.26
2.51
2.53
2.80
3.12
8.05 0.05
7.78 0.01
7.53 0.01
7.43 0.02
7.59 0.02
7.44 0.03
7.64 0.03
7.51 0.01
7.74 0.02
7.78 0.01
7.56 0.03
7.47 0.01
7.52 0.03
7.34 0.01
4.70 0.90
95.3 15.0
31.0 6.0
121 27
89.4 22.0
243 65
>1000
>1000d
19.9 0.9
170 28
211 48
>1000
6217 770
2.95 0.56
22.1 5.5
47.2 4.5
226 58
>1000
5.92 2.25
17.8 1.8
196 63
>1000
29.2 4.2
658 32
133
1341 266
4.2 0.6
7
—
12.5 2.0
—
a
The values are the means SEM from three independent experiments in triplicate (P < 0.001). Individual difference between the various compounds have been examined
using Tukey’s post hoc test (P < 0.001). Difference in the Ki values between the receptors for each compound have been analyzed using the Mann–Whitney U test (P = 0.007,
U = 8.000).
b
Calculated with ACD/Labs 7.0 (Advanced Chemistry Development, Inc., Toronto, ON, Canada).
pKa values of alkylated piperazine nitrogen.
Full Ki not obtained.
c
d

M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
761
F
F
F
Br
Br
N
A
H3CO
16
9a,b-15a,b
G
H
B
O
F
R
R
R
D
C
N
N
N
N
CN
NH
N
N
. HCl
( )n
( )n
( )3
NH2
NH2
37: 2-substituted
38: 3-substituted
17: R= 2-OCH3
18: R= 3-OCH3
19: R= 4-OCH3
20: R= 3-F,5-OCH3
21: R= 2-OH
23: R= 2-OCH3, n= 2
24: R= 3-OCH3, n= 3
25: R= 4-OCH3, n= 3
26: R= 3-F,5-OCH3, n= 3
27: R= 2-OH, n= 3
29: R= 2-OCH3, n= 2
30: R= 3-OCH3, n= 3
31: R= 4-OCH3, n= 3
32: R= 3-F,5-OCH3, n= 3
33: R= 2-OH, n= 3
F
E
O
22: R= 3-OH
28: R= 3-OH, n= 3
34: R= 3-OH, n= 3
F
N
N
( )3
N
Boc
H
35: 2-substituted
36: 3-substituted
Scheme 1. Synthesis of the target compounds 9a,b–15a,b. Reagents and conditions: (A) Sodium methoxide, DMF; (B) piperazine, sodium t-butoxide, dichlorobis(tri-o-
tolylphosphine)palladium, anhydrous toluene; (C)
x-bromoalkylnitrile, K2CO3 (except for 27 and 28), acetonitrile; (D) borane–methyl sulfide complex, anhydrous THF; (E) i:
di-t-butylcarbonate, Et3N, H2O, THF; ii: 2-fluoroethyl mesylate, Cs2CO3, DMF, 65 °C (F) 4 M HCl in dioxane, rt, 3 h. (G) carboxylic acid, 1,10-carbonyldiimidazole, anhydrous
THF, or Et3N, methyl chloroformate; (H) carboxylic acid, PyBOP, N-methylmorpholine, anhydrous CH2Cl2.
which is the less basic compound of the series. Taken together,
these data suggest that, for this class of compounds, clogP values
are reliable for predicting the actual lipophilicity of the target mol-
ecules. Moreover, the pKa values indicate that the piperazine nitro-
gen is not extensively protonated at physiological pH, and this
should be adequately considered in the design of potential PET
tracers with arylpiperazine structure.
the isomers 9a, 11a, and 12a were less marked than that observed
among the isomers 9b, 11b, and 12b. The same affinity trend was
observed for dopamine D2 receptor: Ki values at D2 receptor of
11a,b were approximately 10-fold higher that that of 9a,b, respec-
tively. Shifting of the methoxy group from 3- to 4-position gave
derivatives 12a,b which were devoid of affinity for D2 receptors
(Ki > 1000 nM).
The second set of structural modifications were aimed to in-
clude a fluorine atom into the structure, because fluorine-18 pos-
sess a longer half-life than carbon-11. For this purpose, we
replaced the methoxy group of compounds 9a,b and 11a,b with a
2-fluoroethoxy substituent, leading to compounds 13a,b–14a,b.
This modification affected dopamine D3 affinity in a narrow range,
being 13a equipotent to 9a, and 14a fivefold less potent than 9b.
On the other hand, 13a,b–14a showed higher D2 affinity as com-
pared to their methoxy counterparts. Also, we evaluated fluoroaro-
matic derivatives 15a,b which can be considered formally derived
from 11a,b by introduction of a fluorine on the phenyl ring at-
tached to the piperazine. The 3,5-disubstitution pattern was cho-
sen because it was already reported for other dopamine D3
ligands.30,31 This modification left unchanged D3 affinity for 4-
(1H-imidazol-1-yl)benzamides (11a vs 15a), whereas was detri-
mental for 2,1,3-benzoxadiazole-5-carboxamides (11b vs 15b).
The same trend was shown by D2 affinity data of 15a,b in compar-
ison with 11a,b.
3.2. Structure–affinity relationships
The first set of modifications performed on the reference com-
pounds were shortening of the methylene spacer between the aryl-
carboxamide moiety and the 1-(2-methoxyphenyl)piperazine
(compounds 10a,b) and shifting of the methoxy substituent from
the 2-position to 3- and 4-position of the phenyl attached to the
piperazine ring (compounds 11a,b–12a,b). These structural modi-
fications left unchanged the possibility to access to a labeled com-
pound and also reflected into a slight reduction in lipophilicity.
Shortening of the spacer (9a vs 10a and 9b vs 10b) resulted in a
reduction of D3 affinity of different amplitude: in fact, 10a was
15-fold less potent than 9a, whereas 10b was devoid of D3 affinity.
This modification had a limited impact on D2 receptor affinity,
being 10b only twofold less potent than 9b. Evaluation of the opti-
mal position of the methoxy substituent was carried out because
literature data suggested for similar structure type that a substitu-
ent in 3-position of the phenyl ring could leave unchanged the
affinity for D3 receptor, while reducing the affinity for dopamine
D2 receptor.29 This was not replicated in the present examples:
2-methoxy substituted derivatives were more potent than the cor-
responding 3-methoxy isomers which were more potent than the
4-substituted ones. The difference in D3 receptor affinity among
4. Conclusions
Herein we report an attempt to identify potential PET li-
gands for dopamine D3 receptors. The N-[4-(4-arylpiperazin-1-
yl)butyl]arylcarboxamide scaffold was modified with the aim

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M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
to combine high affinity for D3 receptors, selectivity over D2
receptors, appropriate lipophilicity for high brain uptake and
low non-specific binding, and suitable chemical feature for car-
bon-11 of fluorine-18 labeling. The target compounds 9a,b–
15a,b presented logD7.4 values between 2.22 and 3.12 and, thus,
within the optimal lipophilicity range. As far as the affinity for
D3 receptor is concerned, only compounds 13a, 13b, 14a, and
15a showed good binding properties, being 13a the most potent
of the series. However, the Ki value of 2.95 nM is quite below
the target for optimal Bmax/Kd ratio for D3 receptors. Unfortu-
nately, all the compounds showed modest selectivities over D2
receptors. As pointed out above, the high degree of amino acid
homology between the D2 and D3 dopamine receptor subtypes,
constitutes a formidable challenge in the pursuit to discover
dopamine D3-selective compounds. Within the N-[4-(4-arylpi-
perazin-1-yl)alkyl]arylcarboxamide class, high dopamine D2/D3
selectivity has been achieved with lipophilic arylcarboxylamides
connected through a butyl chain to a 2,3-dichloro- or 2-meth-
oxy-substituted phenylpiperazine as in the case of compounds
1–6 (Table 1). Very recent literature data seem to confirm the
above trend. During the preparation of the present manuscript,
Hocke and coworkers32 have reported on four potential D3 PET
tracer structurally related to compound 13a, where the 4-(1H-
imidazol-1-yl)benzamide moiety was replaced by more lipo-
philic bicyclic systems (i.e., benzothiophene, benzofurane, pyraz-
olo[1,5-a]pyridine, naphthalene). Those compounds were
reported to bind at D3 receptors with subnanomolar affinities
(Ki = 0.12–0.68 nM) and to possess about 100-fold selectivity
over D2 receptor. From these data it became apparent that
maintaining potency and specificity for D3 receptors while opti-
5.1.1. 1-Bromo-3-fluoro-5-methoxybenzene (16)
To an ice-cooled solution of 1-bromo-3,5-difluorobenzene
(3.00 g, 15.6 mmol) in anhydrous DMF (7 mL) was added portion-
wise sodium methoxide (1.69 g, 31.2 mmol). Then, the cooling bath
was removed and the reaction mixture was stirred 24 h at room
temperature. Then, mixture was diluted with CH2Cl2 (40 mL) and
washed with H2O (40 mL). The separated organic layer was dried
(Na2SO4) and concentrated under reduced pressure. The crude res-
idue was chromatographed (petroleum ether as eluent) to give the
title compound as a colorless oil (1.48 g, 46% yield). GC–MS m/z
206 (M++2, 97), 204 (M+, 100), 176 (30), 174 (30), 95 (39). 1H
NMR (CHCl3): d 3.79 (s, 3H), 6.56 (dt, 1H, J = 2.3, 10.5 Hz), 6.82–
6.87 (m, 2H).
5.1.2. 1-(3-Fluoro-5-methoxyphenyl)piperazine (20)
A
mixture of 16 (1.48 g, 7.3 mmol), piperazine (2.49 g,
29.0 mmol), sodium t-butoxide (0.97 g, 10.0 mmol), dichlor-
obis(tri-o-tolylphosphine)palladium (II) (0.16 g, 0.2 mmol) in
anhydrous toluene (30 mL) was warmed at 100 °C overnight. Then,
the mixture was cooled, filtered through Celite, and evaporated to
dryness in vacuo. The residue was taken-up with AcOEt (40 mL)
and washed with H2O (40 mL). The separated organic layer was
dried (Na2SO4) and concentrated under reduced pressure. The
crude residue was chromatographed (CHCl3/MeOH, 9:1) to give
1-arylpiperazine 20 as a pale yellow oil (0.59 g, 39% yield). GC–
MS m/z 211 (M++1, 5), 210 (M+, 34), 168 (100). 1H NMR (CHCl3):
d 1.79 (s, 1H, D2O exchanged), 3.00 (app q, 4H), 3.12 (app q, 4H),
3.75 (s, 3H), 6.12 (dt, 1H, J = 2.3, 10.5 Hz), 6.19–6.25 (m, 2H).
5.2. General procedure for preparation of nitriles 23–29
mizing the physicochemical properties, is still
challenge.
a complex
A stirred suspension of 1-arylpiperazine 17–22 (10 mmol),
x-
bromoalkylnitrile (11 mmol) and K2CO3 (11 mmol) (this reagent
was omitted in the case of arylpiperazines 21 and 22) in acetoni-
trile (50 mL) was refluxed overnight. After cooling, the mixture
was evaporated to dryness and H2O (20 mL) was added to the res-
idue. The aqueous phase was extracted with CH2Cl2 (2ꢀ 30 mL).
The collected organic layers were dried over Na2SO4 and evapo-
rated under reduced pressure. The crude residue was chromato-
graphed as detailed below to yield pure 23–29 as white solids.
5. Experimental
5.1. Chemistry
Column chromatography was performed with 1:30 Merck silica
gel 60A (63–200 m) as the stationary phase. Melting points were
l
determined in open capillaries on a Gallenkamp electrothermal
apparatus. Elemental analyses (C,H,N) were performed on Eurovec-
tor Euro EA 3000 analyzer; the analytical results were within 0.4%
of the theoretical values for the formula given. 1H NMR spectra
were recorded at 300 MHz on a Varian Mercury-VX spectrometer.
All spectra were recorded on free bases. All chemical shift values
are reported in ppm (d). Recording of mass spectra was done on
an HP6890-5973 MSD gas chromatograph/mass spectrometer;
only significant m/z peaks, with their percentage of relative inten-
sity in parentheses, are reported. ESI+/MS/MS analysis were per-
formed with an Agilent 1100 Series LC-MSD trap System VL
workstation. All spectra were in accordance with the assigned
structures. The purity of new compounds that were essential to
the conclusions drawn in the text was determined by HPLC on a
Perkin-Elmer series 200 LC instrument using a Phenomenex Gem-
5.2.1. 4-[4-(4-Methoxyphenyl)piperazin-1-yl)butanenitrile (25)
Eluted with CHCl3/AcOEt, 1:1. 83% Yield. GC–MS m/z 260 (M++1,
18), 259 (M+, 100), 219 (36). 1H NMR (CHCl3): d 1.87 (quintet, 2H,
J = 6.9 Hz), 2.46 (t, 2H, J = 7.2 Hz), 2.53 (t, 2H, J = 6.9 Hz), 2.62 (app
t, 4H), 3.10 (app t, 4H), 3.77 (s, 3H), 6.82–6.92 (m, 4H).
5.2.2. 4-[4-(3-Fluoro-5-methoxyphenyl)piperazin-1-
yl)butanenitrile (26)
Eluted with CHCl3/AcOEt, 1:1. 95% Yield. GC–MS m/z 278 (M++1,
18), 277 (M+, 100), 237 (55), 223 (49), 123 (50). 1H NMR (CHCl3): d
1.85 (quintet, 2H, J = 6.9 Hz), 2.45 (t, 2H, J = 7.2 Hz), 2.50 (t, 2H,
J = 6.7 Hz), 2.56 (app t, 4H), 3.17 (app t, 4H), 3.76 (s, 3H), 6.12
(dt, 1H, J = 2.1, 5.2 Hz), 6.18–6.20 (m, 1H), 6.24 (t, 1H, J = 2.1 Hz).
ini RP-18 column, (250 ꢀ 4.6 mm, 5
lm particle size) and equipped
5.2.3. 4-[4-(2-Hydroxyphenyl)piperazin-1-yl)butanenitrile (27)
Eluted with CHCl3/MeOH, 19:1. 76% Yield. GC–MS m/z 246
(M++1, 5), 245 (M+, 32), 205 (29), 148 (100). 1H NMR (CHCl3): d
1.86 (quintet, 2H, J = 6.9 Hz), 2.47 (t, 2H, J = 7.0 Hz), 2.54 (t, 2H,
J = 6.7 Hz), 2.58 (app t, 4H), 2.90 (app t, 4H), 6.83–6.89 (m, 1H),
6.94 (dd, 1H J = 1.7, 8.8 Hz), 7.02 (br s, 1H, D2O exchanged), 7.05–
7.15 (m, 1H), 7.17 (dd, 1H, J = 1.7, 8.5 Hz).
with a Perkin-Elmer 785A UV/VIS detector setting k = 254 nm.
Compounds 9a,b–15a,b were eluted with CH3OH/H2O/Et3N,
4:1:0.01, v/v at a flow rate of 0.8 mL/min. When necessary, a stan-
dard procedure was used to transform final compounds into their
hydrochloride salts. The following compounds were synthesized
according to published procedures: 3-[4-(2-methoxyphenyl)pip-
erazin-1-yl)propanenitrile (23),33 4-[4-(3-methoxyphenyl)pipera-
zin-1-yl)butanenitrile
(24),34
3-(2-methoxyphenyl)-1-
5.2.4. 4-[4-(3-Hydroxyphenyl)piperazin-1-yl)butanenitrile (28)
Eluted with CHCl3/MeOH, 19:1. 44% Yield. GC–MS m/z 246
(M++1, 17), 245 (M+, 100), 205 (52), 191 (38). 1H NMR (CHCl3): d
piperazinepropanamine (29),33 4-(3-methoxyphenyl)-1-piperazi-
nebutanamine (30).34

M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
763
1.86 (quintet, 2H, J = 6.9 Hz), 2.45 (t, 2H, J = 7.2 Hz), 2.51 (t, 2H,
J = 6.7 Hz), 2.58 (app t, 4H), 3.17 (app t, 4H), 5.36 (br s, 1H, D2O ex-
changed), 6.29–6.33 (m, 1H), 6.37 (t, 1H, J = 2.3 Hz), 6.49 (dd, 1H,
J = 1.9, 8.2 Hz), 7.10 (t, 1H, J = 8.1 Hz).
the solvent in vacuo gave a crude residue which was chromato-
graphed (CHCl3/MeOH, 19:1) to afford pure 35 as a pale yellow
oil (0.62 g, 65% yield). ESI+/MS m/z 396 (MH+). ESI+/MS/MS m/z
296 (100), 279 (33), 225 (20). 1H NMR (CHCl3): d 1.43 (s, 9H),
1.54–1.56 (m, 4H), 2.39 (app t, 2H), 2.95 (br s, 4H), 3.04 (br s,
2H), 3.15 (app t, 4H), 4.19–4.32 (m, 2H), 4.68–4.87 (m, 2H),
5.30 (br s, 1H), 6.83–6.88 (m, 1H), 6.91–6.99 (m, 2H), 7.02–7.09
(m, 1H).
5.3. General procedure for preparation of amines 31–34
Borane–methyl sulfide complex, as 10.0 M BH3 in excess methyl
sulfide (1.2 mL, 12.0 mmol) was dropped into an ice-cooled solu-
tion of nitrile 25–28 (4.0 mmol) in anhydrous THF (10 mL), under
stirring. After being refluxed for 1 h, the reaction mixture was
cooled at ꢁ10 °C and MeOH was added very carefully dropwise un-
til gas evolution ceased. The mixture was treated with 3 N HCl
(5 mL) and was refluxed for 1 h. After cooling, the mixture was alk-
alized with 3 N NaOH and extracted with CH2Cl2 (2ꢀ 50 mL). The
collected organic layers were dried over Na2SO4 and the solvent
was evaporated under reduced pressure to give the amines 31–
34 as a white semisolids which were used for the next step without
further purification.
5.3.6. N-t-Butoxycarbonyl-4-(3-(2-fluoroethoxy)phenyl)-1-
piperazinebutanamine (36)
As above, starting from amine 34 the title compound was ob-
tained in 65% yield. ESI+/MS m/z 396 (MH+). ESI+/MS/MS m/z 296
(100), 279 (34). 1H NMR (CHCl3): d 1.42 (s, 9H), 1.54–1.56 (m,
4H), 2.36 (t, 2H, J = 6.9 Hz), 2.58 (app t, 4H), 3.12–3.16 (m, 2H),
3.21 (app t, 4H), 4.13–4.52 (m, 2H), 4.58–4.83 (m, 2H), 5.23 (br s,
1H), 6.40 (dd, 1H, J = 2.2, 8.0 Hz), 6.50 (t, 1H, J = 2.3 Hz), 6.54–
6.58 (m, 1H), 7.16 (t, 1H, J = 8.3 Hz).
5.3.7. 4-(2-(2-Fluoroethoxy)phenyl)-1-piperazinebutanamine
(37)
5.3.1. 4-(4-Methoxyphenyl)-1-piperazinebutanamine (31)
Quantitative yield. GC–MS m/z 264 (M++1, 13), 263 (M+, 65),
248 (32), 205 (59), 163 (93), 127 (100). 1H NMR (CHCl3): d 1.44–
1.64 (m, 4H), 2.25 (br s, 2H, D2O exchanged), 2.40 (t, 2H,
J = 7.2 Hz), 2.62 (app t, 4H), 2.74 (t, 2H, J = 6.6 Hz), 3.10 (app t,
4H), 3.75 (s, 3H), 6.81–6.97 (m, 4H).
A mixture of 35 (0.28 g, 0.7 mmol) and 4 M HCl in dioxane
(20 mL) was stirred at room temperature 3 h. Then, the solvent
was removed under reduced pressure. The obtained hydrochlo-
ride salt was dried in vacuo and used for the next step with-
out any further purification. ESI+/MS m/z 296 (MH+). ESI+/MS/
MS m/z 279 (100), 225 (60). 1H NMR (free base, CHCl3): d
1.43–1.62 (m, 6H, 2H D2O exchanged), 2.41 (t, 2H, J = 7.4 Hz),
2.64 (br s, 4H), 2.72 (t, 2H, J = 6.7 Hz), 3.13 (app t, 4H),
4.21–4.31 (m, 2H), 4.68–4.88 (m, 2H), 6.83–6.88 (m, 1H),
6.94–6.98 (m, 3H).
5.3.2. 4-(3-Fluoro-5-methoxyphenyl)-1-piperazinebutanamine
(32)
84% Yield. GC–MS m/z 281 (M+, 2), 208 (21), 168 (69), 91 (100).
1H NMR (CHCl3): d 1.42–1.60 (m, 4H), 2.00 (br s, 2H, D2O ex-
changed), 2.38 (t, 2H, J = 6.9 Hz), 2.56 (app t, 4H), 2.75 (t, 2H,
J = 6.2 Hz), 3.17 (app t, 4H), 3.75 (s, 3H), 6.10 (d, 1H, J = 10.5 Hz),
6.19 (s, 1H), 6.21 (d, 1H, J = 12.7 Hz).
5.3.8. 4-(3-(2-Fluoroethoxy)phenyl)-1-piperazinebutanamine
(38)
Amine 38 hydrochloride was obtained as above starting from
36. ESI+/MS m/z 296 (MH+). ESI+/MS/MS m/z 279 (100), 225 (29).
1H NMR (free base, CHCl3): d 1.43–1.62 (m, 4H), 1.69 (s, 2H, D2O
exchanged), 2.40 (t, 2H, J = 7.4 Hz), 2.59 (app t, 4H), 2.73 (t, 2H,
J = 6.7 Hz), 3.20 (app t, 4H), 4.13–4.26 (m, 2H), 4.64–4.83 (m, 2H),
6.40 (dd, 1H, J = 2.2, 7.7 Hz), 6.50 (t, 1H, J = 2.3 Hz), 6.55–6.58 (m,
1H), 7.16 (t, 1H, J = 8.2 Hz).
5.3.3. 2-[4-(4-Aminobutyl)-1-piperazinyl]phenol (33)
44% Yield. GC–MS m/z 250 (M++1, 3), 249 (M+, 10), 191 (52), 148
(48), 134 (69), 127 (100). 1H NMR (CHCl3): d 1.48–1.63 (m, 4H),
2.43 (t, 2H, J = 6.2 Hz), 2.62 (br s, 7H), 2.74 (br s, 2H), 2.91 (br s,
4H), 6.85 (dt, 1H, J = 1.3, 7.6 Hz), 6.92 (dd, 1H, J = 1.2, 8.1 Hz),
7.06 (dt, 1H, J = 1.3, 7.6 Hz), 7.17 (dd, 1H, J = 1.1, 6.6 Hz).
5.3.4. 3-[4-(4-Aminobutyl)-1-piperazinyl]phenol (34)
46% Yield. ESI+/MS m/z 250 (MH+). ESI+/MS/MS m/z 233 (100),
179 (28). 1H NMR (CHCl3): d 1.48–1.62 (m, 4H), 2.39 (t, 2H,
J = 7.3 Hz), 2.57 (app t, 4H), 2.75 (t, 2H, J = 6.6 Hz), 3.02 (br s, 3H,
D2O exchanged), 3.15 (app t, 4H), 6.29 (dd, 1H, J = 1.6, 7.8 Hz),
6.36 (t, 1H, J = 2.2 Hz), 6.44 (dd, 1H, J = 1.9, 8.3 Hz), 7.06 (t, 1H,
J = 8.1 Hz).
5.4. General procedure for the synthesis of carboxamides 10a,b,
11a, 12a,b, and 15a,b
A mixture of the appropriate carboxylic acid (0.48 mmol) and
1,10-carbonyldiimidazole (0.50 mmol) in 10 mL of anhydrous THF
was stirred for 8 h. A solution of amine 29–31 (0.48 mmol) in
10 mL of anhydrous THF was added and the mixture was stirred
until the carboxylic acid disappeared (TLC). The reaction mixture
was partitioned between AcOEt (20 mL) and H2O (20 mL). The sep-
arated organic layer was washed with aqueous Na2CO3 solution
(20 mL), dried (Na2SO4) and concentrated in vacuo. The crude res-
idue was chromatographed with CHCl3/CH3OH, 19:1, to afford the
pure arylcarboxamide.
5.3.5. N-t-Butoxycarbonyl-4-(2-(2-fluoroethoxy)phenyl)-1-
piperazinebutanamine (35)
Amine 33 (0.71 g, 2.9 mmol) was solubilized in a mixture of H2O
(10 mL) and THF (30 mL). Et3N (0.4 mL, 2.9 mmol) and di-t-butyl-
carbonate (0.62 g, 2.9 mmol) were added to the solution. The mix-
ture was stirred at room temperature overnight. Then, THF was
evaporated in vacuo and the aqueous residue was extracted with
AcOEt (3ꢀ 20 mL). The organic phases were collected, dried over
Na2SO4 and concentrated under reduced pressure. The crude resi-
due was chromatographed (CHCl3/MeOH, 19:1 as eluent) to give
0.85 g of N-BOC-33 as white semisolid. A mixture of N-BOC-33
(0.85 g, 2.4 mmol), 2-fluoroethyl mesylate (0.53 g, 4.9 mmol), and
Cs2CO3 (0.48 g, 1.5 mmol) in DMF was warmed at 65 °C for 3 h.
Then, the solvent was distilled off and the residue was taken-up
with AcOEt (30 mL). The organic phase was washed first with
H2O (20 mL), with brine, then dried over Na2SO4. Evaporation of
5.4.1. N-[4-[3-(3-Methoxyphenyl)piperazin-1-yl]propyl]-4-(1H-
imidazol-1-yl)benzamide (10a)
35% Yield. GC–MS m/z 420 (M++1, 11), 419 (M+, 47), 404 (30),
257 (25), 205 (100). 1H NMR (CHCl3): d 1.86–1.92 (m, 2H), 2.73
(t, 2H, J = 5.6 Hz), 2.79 (br s, 4H), 3.12 (br s, 4H), 3.63 (q, 2H,
J = 5.4 Hz), 3.86 (s, 3H), 6.84–6.94 (m, 3H), 7.02–7.07 (m, 1H),
7.22 (app s, 1H), 7.27–7.29 (m, 1H), 7.40–7.44 (m, 2H), 7.86 (s,
1H), 7.99 (d, 2H, J = 8.5 Hz), 8.67 (br t, 1H). The hydrochloride salt
melted at 187–189 °C (from MeOH/Et2O), Anal. (C24H29N5O2ꢂ
3HClꢂ2H2O) C, H, N.

764
M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
5.4.2. N-[4-[3-(2-Methoxyphenyl)piperazin-1-yl]propyl]-2,1,3-
benzoxadiazole-5-carboxamide (10b)
morpholine (2.6 mmol) in 10 mL of anhydrous CH2Cl2 was stirred
overnight. Then, the mixture was washed with saturated aqueous
NaHCO3 (10 mL). The organic layer was separated, dried (Na2SO4)
and concentrated in vacuo. The crude residue was chromato-
graphed with CHCl3/CH3OH, 19:1, to afford the pure
arylcarboxamide.
41% Yield. ESI+/MS m/z 396 (MH+). ESI+/MS/MS m/z 204 (100),
193 (87), 147 (44). 1H NMR (CDCl3): d 1.87–1.95 (m, 2H), 2.76 (t,
2H, J = 5.6 Hz), 2.82 (br s, 4H), 3.12 (br s, 4H), 3.65 (q, 2H,
J = 5.5 Hz), 3.85 (s, 3H), 6.85 (d, 2H, J = 7.4 Hz), 6.90–6.96 (m, 1H),
7.00–7.06 (m, 1H), 7.84–7.94 (m, 2H), 8.35 (s, 1H), 9.03 (br s, 1H,
D2O exchanged). The hydrochloride salt melted at 205–207 °C
(from MeOH/Et2O), Anal. (C21H25N5O3ꢂ2HClꢂ0.2H2O) C, H, N.
5.5.1. N-[4-[4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-yl]butyl]-
4-(1H-imidazol-1-yl)benzamide (13a)
57% Yield. ESI+/MS m/z 466 (MH+). ESI+/MS/MS m/z 279 (36),
242 (90), 225 (29), 171 (100). 1H NMR (CHCl3): d 1.72–1.80 (m,
4H), 2.64 (t, 2H, J = 6.8 Hz), 2.83 (br s, 4H), 3.18 (br s, 4H), 3.46–
3.55 (m, 2H), 4.18–4.30 (m, 2H), 4.67–4.86 (m, 2H), 6.84–6.89
(m, 2H), 6.91–7.02 (m, 2H), 7.22 (s, 1H), 7.25 (br s, 1H), 7.29–
7.30 (m, 1H), 7.43 (d, 2H, J= 8.5 Hz), 7.87 (br s, 1H), 7.96 (d, 2H,
J = 8.5 Hz). The free base melted at 92–93 °C (from CHCl3/n-hex-
ane), Anal. (C26H32FN5O2) C, H, N.
5.4.3. N-[4-[4-(3-Methoxyphenyl)piperazin-1-yl]butyl]-4-(1H-
imidazol-1-yl)benzamide (11a)
21% Yield. ESI+/MS m/z 434 (MH+). ESI+/MS/MS m/z 247 (30),
242 (69), 171 (100). 1H NMR (CHCl3): d 1.67–1.75 (m, 4H), 2.49
(t, 2H, J = 6.7 Hz), 2.63 (app t, 4H), 3.17 (app t, 4H), 3.47–3.54 (m,
2H), 3.77 (s, 3H), 6.41 (app t, 2H), 6.50 (dd, 1H, J = 2.2, 8.2 Hz),
6.94 (br s, 1H), 7.13–7.18 (m, 1H), 7.22 (app s, 1H), 7.28 (t, 1H,
J = 1.2 Hz), 7.39–7.44 (m, 2H), 7.86–7.91 (m, 3H). The free base
melted at 124–126 °C (from CHCl3/n-hexane), Anal. (C25H31N5O2)
C, H, N.
5.5.2. N-[4-[4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-yl]butyl]-
2,1,3-benzoxadiazole-5-carboxamide (13b)
38% Yield. ESI+/MS m/z 442 (MH+). ESI+/MS/MS m/z 279 (40),
225 (90), 218 (90), 182 (33), 147 (100). 1H NMR (CHCl3): d 1.74–
1.79 (m, 4H), 2.52 (t, 2H, J = 6.6 Hz), 2.68 (br s, 4H), 3.07 (br s,
4H), 3.52 (q, 2H, J = 5.6 Hz), 4.17–4.29 (m, 2H), 4.66–4.85 (m,
2H), 6.75 (m, 2H), 6.89–7.00 (m, 2H), 7.47 (br t, 1H), 7.83–7.91
(m, 2H), 8.21 (t, 1H, J = 1.1 Hz). The free base melted at 104–
107 °C (from CHCl3/n-hexane), Anal. (C23H28FN5O3) C, H, N.
5.4.4. N-[4-[4-(4-Methoxyphenyl)piperazin-1-yl]butyl]-4-(1H-
imidazol-1-yl)benzamide (12a)
35% Yield. ESI+/MS m/z 434 (MH+). ESI+/MS/MS m/z 247 (24),
242 (63), 171 (100). 1H NMR (CHCl3): d 1.68–1.73 (m, 4H), 2.51
(t, 2H, J = 6.7 Hz), 2.67 (app t, 4H), 3.08 (app t, 4H), 3.47–3.57 (m,
2H), 3.75 (s, 3H), 6.79–6.87 (m, 4H), 7.02 (br t, 1H), 7.22 (app s,
1H), 7.28 (t, 1H, J = 1.2 Hz), 7.41–7.44 (m, 2H), 7.87–7.92 (m, 3H).
The free base melted at 144–146 °C (from CHCl3/n-hexane), Anal.
(C25H31N5O2) C, H, N.
5.5.3. N-[4-[4-(3-(2-Fluoroethoxy)phenyl)piperazin-1-yl]butyl]-
4-(1H-imidazol-1-yl)benzamide (14a)
48% Yield. ESI+/MS m/z 466 (MH+). ESI+/MS/MS m/z 279 (30),
242 (71), 171 (100). 1H NMR (CHCl3): d 1.68–1.75 (m, 4H), 2.51
(t, 2H, J = 6.7 Hz), 2.65 (app t, 4H), 3.18 (app t, 4H), 3.51 (q, 2H,
J = 6.1 Hz), 4.12–4.24 (m, 2H), 4.64–4.82 (m, 2H), 6.41 (dd, 1H,
J = 2.3, 8.1 Hz), 6.46 (t, 1H, J = 2.3 Hz), 6.53 (dd, 1H, J = 2.3,
8.2 Hz), 6.97 (br t, 1H), 7.16 (t, 1H, J = 8.1 Hz), 7.21 (t, 1H,
J = 1.1 Hz), 7.26–7.29 (m, 1H), 7.40–7.45 (m, 2H), 7.86–7.91 (m,
3H). The free base melted at 118–120 °C (from CHCl3/n-hexane),
Anal. (C26H32FN5O2) C, H, N.
5.4.5. N-[4-[4-(4-Methoxyphenyl)piperazin-1-yl]butyl]-2,1,3-
benzoxadiazole-5-carboxamide (12b)
54% Yield. ESI+/MS m/z 410 (MH+). ESI+/MS/MS m/z 247 (34),
218 (73), 193 (56), 176 (30), 147 (100). 1H NMR (CDCl3): d 1.69–
1.77 (m, 4H), 2.50 (t, 2H, J = 6.3 Hz), 2.64 (app t, 4H), 3.04 (app t,
4H), 3.52 (q, 2H, J = 5.9 Hz), 3.76 (s, 3H), 6.81 (s, 4H), 7.30 (br t,
1H), 7.85 (q, 2H, J = 8.9 Hz), 8.18 (s, 1H). The free base melted at
135–137 °C (from CHCl3/n-hexane), Anal. (C22H27N5O3) C, H, N.
5.4.6. N-[4-[4-(3-Fluoro-5-methoxyphenyl)piperazin-1-
yl]butyl]-4-(1H-imidazol-1-yl)benzamide (15a)
5.5.4. N-[4-[4-(3-(2-Fluoroethoxy)phenyl)piperazin-1-yl]butyl]-
2,1,3-benzoxadiazole-5-carboxamide (14b)
69% Yield. ESI+/MS m/z 452 (MH+). ESI+/MS/MS m/z 265 (25),
242 (72), 171 (100). 1H NMR (CHCl3): d 1.62–1.77 (m, 4H), 2.48
(t, 2H, J = 6.9 Hz), 2.60 (app t, 4H), 3.15 (app t, 4H), 3.50 (q, 2H,
J = 6.1 Hz), 3.74 (s, 3H), 6.09–6.21 (m, 3H), 6.89 (br t, 1H), 7.21 (t,
1H, J = 1.1 Hz), 7.29 (t, 1H, J = 1.4 Hz), 7.40–7.44 (m, 2H), 7.87–
7.92 (m, 3H). The free base melted at 144–146 °C (from CHCl3/n-
hexane), Anal. (C25H30FN5O2) C, H, N.
70% Yield. ESI+/MS m/z 442 (MH+). ESI+/MS/MS m/z 279 (41),
225 (53), 218 (82), 176 (20), 147 (100). 1H NMR (CDCl3): d 1.67–
1.76 (m, 4H), 2.48 (t, 2H, J = 6.7 Hz), 2.61 (app t, 4H), 3.13 (br s,
4H), 3.51 (q, 2H, J = 6.1 Hz), 4.13–4.25 (m, 2H), 4.65–4.84 (m,
2H), 6.38–6.44 (m, 2H), 6.47–6.51 (m, 1H), 7.14 (t, 1H, J = 8.1 Hz),
7.43 (br t, 1H, D2O exchanged), 7.86 (t, 2H, J = 1.1 Hz), 8.23 (t, 1H,
J = 1.2 Hz). The free base melted at 83–87 °C (from CHCl3/n-hex-
ane), Anal. (C23H28FN5O3) C, H, N.
5.4.7. N-[4-[4-(3-Fluoro-5-methoxyphenyl)piperazin-1-
yl]butyl]-2,1,3-benzoxadiazole-5-carboxamide (15b)
57% Yield. ESI+/MS m/z 428 (MH+). ESI+/MS/MS m/z 265 (34),
218 (86), 211 (31), 176 (21), 147 (100). 1H NMR (CDCl3): d 1.67–
1.77 (m, 4H), 2.52 (t, 2H, J = 6.9 Hz), 2.63 (app t, 4H), 3.17 (app t,
4H), 3.53 (q, 2H, J = 6.1 Hz), 3.76 (s, 3H), 6.10–6.19 (m, 3H), 7.15
(br t, 1H), 7.82–7.91 (m, 2H), 8.20 (t, 1H, J = 1.1 Hz). The free base
melted at 129–131 °C (from CHCl3/n-hexane), Anal. (C22H26FN5O3)
C, H, N.
5.5.5. N-[4-[4-(3-Methoxyphenyl)piperazin-1-yl]butyl]-2,1,3-
benzoxadiazole-5-carboxamide (11b)
A mixture of carboxylic acid (0.25 g, 1.5 mmol) in CHCl3 (10 mL)
and triethylamine (0.24 mL, 1.7 mmol) was stirred at room tem-
perature for 15 min. After cooling at ꢁ10 °C methyl chloroformate
(0.13 mL, 1.7 mmol) was added and the mixture reacted at the
same temperature for 1 h. Then a solution of 4-(3-methoxy-
phenyl)-1-piperazinebutanamine (30) (0.44 g, 1.7 mmol) in CHCl3
was dropped into and the resulting mixture was kept at -10 °C to
ꢁ5 °C for 1 h. After stirring overnight at room temperature, the
reaction mixture was washed with 5% aqueous NaOH, with water
and dried over Na2SO4. Evaporation of the solvent in vacuo affor-
ded the crude product which was purified by column chromatog-
raphy (eluent CHCl3/MeOH, 19:1) to give 11b as a yellow solid
(0.23 g, 37% yield). GC–MS m/z 410 (M++1, 16), 409 (M+, 55), 394
5.5. General procedure for the synthesis of carboxamides 13a,b
and 14a,b
A mixture of 37 or 38 hydrochloride (0.65 mmol), appropriate
carboxylic acid (0.65 mmol), benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate (0.97 mmol), and N-methyl

M. Leopoldo et al. / Bioorg. Med. Chem. 17 (2009) 758–766
765
(24), 205 (100). 1H NMR (CDCl3): d 1.68–1.77 (m, 4H), 2.50 (t, 2H,
J = 6.7 Hz), 2.64 (app t, 4H), 3.15 (app t, 4H), 3.52 (q, 2H, J = 6.1 Hz),
3.78 (s, 3H), 6.38–6.48 (m, 3H), 7.15 (t, 1H, J = 8.1 Hz), 7.23 (br t,
1H), 7.81–7.90 (m, 2H), 8.19 (t, 1H, J = 1.1 Hz). The free base melted
at 119–121 °C (from CHCl3/n-hexane), Anal. (C22H27N5O3) C, H, N.
six to nine concentrations of drug solution in a final volume of
500 L. The samples were incubated for 120 min at 25 °C, then
the incubation was stopped by rapid filtration through Whatman
GF/C glass fiber filters (presoaked in 0.5% polyethylenimine for
60 min). The filters were washed 3 ꢀ 1 mL of ice-cold 50 mM Tris,
0.9% NaCl, pH 7.4. Non-specific binding was determined in the
l
5.6. Lipophilicity data
presence of 10 lM haloperidol. The radioactivity bound to the fil-
ters was measured by liquid scintillation using LS6500 Multi-Pur-
Lipophilicity data of compounds 9a,b–15a,b were obtained by
the pH metric technique using a GlpKa apparatus (Sirius Analytical
Instruments Ltd, Forrest Row, East Sussex, United Kingdom) as de-
scribed elsewhere.35–38 The low aqueous solubility of the investi-
gated compounds required pKa measurements to be performed in
the presence of methanol as co-solvent. Three separate 20 mL-
semiaqueous solutions of approximately 5 ꢀ 10ꢁ5 M, in 20–40%
w/w of MeOH, were initially acidified with 0.5 M HCl to pH 3.0.
The solutions were then titrated with 0.5 M KOH to pH 11. The ini-
tial estimates of the psKa values, which are the apparent ionization
constants in the mixed solvent, were obtained by Bjerrum plots.
These values were then refined by a weighted nonlinear least-
squares procedure (Refinement Pro 1.0 software) to create a multi-
set, where the refined values were extrapolated to zero co-solvent
concentration using the Yasuda–Shedlovsky equation.39 To obtain
logP data, at least three separate titrations were performed on each
compound, on approximately 5 ꢀ 10ꢁ5 M, in the presence of differ-
ent volumes of n-octanol (ratios ranging from 0.005 to 1). The bi-
phasic solutions were initially acidified to pH 3.0 with 0.5 M HCl
and then titrated with 0.5 M KOH to pH 11. The obtained data were
refined as described above. The logP values were obtained by the
Multiset approach, as described elsewhere.36,37 All titrations were
carried out at 25 0.1 °C under an inert nitrogen gas atmosphere
to exclude CO2.
pose scintillation Counter, Beckman.
5.7.4. Statistical analysis
The inhibition curves on the different binding sites of the com-
pounds reported in Table 2 were analyzed by nonlinear curve fit-
ting utilizing the GraphPad Prism program.41 The value for the
inhibition constant, Ki, was calculated by using the Cheng–Prusoff
equation.42
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.11.044.
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